中华鲟子一代配子质量及其子二代生长特征分析

通过测定卵的直径、卵粒质量、受精率和孵化率系列指标以及精子的激活率、快速运动时间和寿命系列指标对人工驯养子一代中华鲟配子进行质量评价,并通过该批受精卵孵出的子二代系列生长指标进行其发育状况分析。结果显示,子一代所产卵子形态饱满,卵径平均3.71 mm,小于自然繁殖群体。与历年在本基地养殖的自然繁殖群体统计资料相比,其受精率(60.1%)和孵化率(36.8%)处在中等偏下的水平。2012年和2013年的精子活力监测结果发现,子一代精子有效运动时间(漩涡运动+快速运动)均较野生群体高。表明子一代卵子质量较野生群体有所降低,而精子活力则较野生群体高。与历年该基地集约化养殖系统培育出的子一代中华鲟相比,子二代的生长表现出显著的阶段差异性,其中仔鱼阶段生长较慢,稚鱼和幼鱼阶段生长优势则较明显。研究表明,促进人工驯养条件下的子一代后备亲鱼性腺发育完善,以获得较高质量的配子和子二代,对防止中华鲟种质退化至关重要。     

我国主要淡水养殖鱼类精液的生物学特性

测定了青鱼、草鱼、鲢、鳙、团头鲂等鱼类的精液浓度,精子密度,精子的形态和大小,精子的活力和寿命,精子的 pH 值,精子数量与受精率的关系.     

PI/Rh123双染和流式细胞术作用下不同盐度对葡萄牙牡蛎精子质量及受精的影响

以葡萄牙牡蛎精子为对象,采用碘化丙啶(PI)/罗丹明123(Rh123)双染与流式细胞术(FCM),研究了不同盐度胁迫(10、15、20、25、30、35)对精子质量的影响,并对受胁迫精子进行授精实验。结果显示,随着盐度升高,精子的存活率、运动时间和活力先升高后降低;葡萄牙牡蛎精子的适宜盐度为20～35,活力较高的盐度范围为25～35;通过双染和FCM检测了精子质膜完整性和线粒体活性,发现盐度胁迫先对精子质膜造成损伤,后对线粒体活性造成伤害;低盐胁迫(10、15)对精子损伤严重,胁迫15 min时质膜受损比例高达67.26%±2.35%。人工授精结果显示,低盐胁迫下精子受精率和卵裂率显著降低,且卵裂阶段出现畸形及分裂停止等现象,表明精子质量不仅严重影响了受精过程,还对卵裂造成一定影响。本实验不仅探究了PI/Rh123双染和FCM检测牡蛎精子质膜完整性和线粒体活性的可行性,还结合显微观察全面评价了精子质量,为牡蛎精子质量的研究提供了理论基础。     

双斑东方鲀精子冷冻保存方法探究

双斑东方鲀(Takifugu bimaculatus)在福建已形成较大规模的养殖产业,但其精卵成熟同步率低、自然交配受精率低等繁殖特点已成为养殖规模化发展的最大制约因素。而精子超低温冷冻保存技术作为直接保存细胞遗传物质的一种有效方法,为双斑东方鲀规模化人工繁育问题的解决提供了方案。研究以复苏后的精子活力为选择指标,开展双斑东方鲀精子冷冻保存方法的研究,对抗冻剂、基础液、精子稀释浓度和降温程序等几个关键影响因素进行了筛选,获得了双斑东方鲀精子的最适冷冻方法:以含5%二甲亚砜(DMSO)的MPRS溶液与精子按1∶1比例稀释,样品在4℃冰箱中平衡30 min,液氮上方10 cm处熏蒸5 min,再下降到5 cm处熏蒸5 min,之后投入液氮。按此方法保存的双斑东方鲀精子在解冻激活后,其活力可达(83±3)%,与相应卵子受精,可得到平均70%的孵化率,可以满足双斑东方鲀规模化人工繁育的需求。研究结果也可为其他东方鲀鱼类的相关研究提供参考。     

温度对海捕野生大黄鱼精子活力的影响

给7尾体质量325~520g的成熟海捕野生大黄鱼人工催产后采精液,在20℃(室温)和4℃(冰箱)下保存0、1、3、6、12、24、36h,以砂滤海水为激活剂,观察大黄鱼精子活力,以精子的激烈运动、摇尾运动、缓慢运动和寿命作为评价指标。试验结果表明,4℃和20℃下随保存时间的延长,精子活力逐渐减弱;4℃下保存24h、20℃下保存12h,精子仍具有激烈运动的能力;4℃下保存的3个运动阶段和寿命较20℃下均有所延长,4℃保存大黄鱼精子更佳。     

人工养殖西伯利亚鲟精子超低温冷冻保存研究

研究了人工养殖西伯利亚鲟精子的生物学特征及超低温冷冻保存方法。西伯利亚鲟的产精量为113.67±39.86 ml,精子密度为(6.49±3.10)×108/ml,精子活力为(85.4±9.5)%,精子寿命为353±23 s。精子密度与精子快速运动时间、精子寿命之间均存在线性相关,用方程分别表示为:y=1.0384x+1.5089(R2=0.7325);y=2.9069x+74.289(R2=0.6967)。结果表明精子密度可作为一项精子质量评价的标准。通过比较西伯利亚鲟精子在不同稀释液、不同抗冻剂和抗冻剂浓度、降温速率、解冻温度下的保存效果,结果表明:配方2作为稀释液,18%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存精子,40℃水浴解冻取得最好的冻后活力,解冻后活力为(51.8±5.8)%。西伯利亚鲟授精的最佳精卵比为106∶1。在此精卵比下用冻精授精分别得到了(72.3±3)%的受精率和(52.9±4.1)%的孵化率,其中受精率与鲜精没有显著性差异,孵化率与鲜精有显著差异(P<0.05)。     

高白鲑精子活力观察

精子活力泛指精子群体的运动状况,包括精子的激活与抑制、运动精子占精子总数的比例（激活率）、精子运动激烈程度及运动时间等,反映了精子的质量。高白鲑属鲑形目、鲑科、白鲑属,2003年由黑龙江省水产技术推广总站从新疆赛里木湖引进,投放到五大连池山口水库,     

不同离子浓度、温度、pH对拉氏(岁)精子活力的影响

为探究拉氏(岁)精子的生理特性和提高拉氏(岁)人工繁殖受精率,探明金属离子浓度、温度、pH等因子变化对拉氏(岁)精子活力的影响.取10尾拉氏(岁)雄亲鱼(体质量80.0～100.0 g,体长10.0～20.0 cm),测定其在不同离子[Na+、K+、Ca2+、葡萄糖、Tris缓冲剂(C4 H11 NO3)]、不同温度[...     

长期超低温冷冻保存对鞍带石斑鱼精子超微结构及酶活性的影响

【目的】本文旨在探究长期超低温冷冻保存中鞍带石斑鱼精子质膜、活力、超微结构及酶活性的变化，为阐明影响鞍带石斑鱼精子冷冻保存质量的相关机制提供理论依据。【方法】采集2022年鞍带石斑鱼鲜精及储存时间分别为23、49、61个月冷冻保存精液，用伊红-苯胺黑染色方法检测精子质膜完整性；用计算机辅助精子分析仪（CASA）检测精子运动参数；测量精浆和精子中琥珀酸脱氢酶（SDH）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）、总超氧化物歧化酶（T-SOD）、谷胱甘肽过氧化物酶（GSH-PX）和肌酸激酶（CK）共六种酶活性的变化及三磷酸腺苷（ATP）浓度变化；用扫描电镜和透射电镜观察鲜精和冻精超微结构。【结果】伊红-苯胺黑染色检测结果发现，鲜精质膜完整性最高为83.43±2.73 %，经过超低温冷冻后，精子质膜完整性显著降低（P<0.05），且随着冷冻保存时间的延长而逐渐降低。CASA结果显示鲜精活力最高为90.47±3.34 %，经过超低温冷冻后精子活力显著降低（P<0.05），但长期保存23-61个月精子活力无显著性差异，精子活力保持在63.95±3.66 %-68.58±2.73 %，具有稳定的活力，且鲜精与冻精之间精子平均直线运动速度（VSL）、平均曲线运动速度（VCL）和平均路径速度（VAP）均没有显著差异（P>0.05）。精子超微结构显示，鲜精形态结构正常、线粒体排列结构规则、形态大小正常。经过超低温冷冻保存后，精子形态结构损伤明显，表现为精子头部质膜破损、细胞质外漏、细胞核膜破损、尾部鞭毛断裂或脱落等损伤；鞍带石斑鱼精浆和精子超低温冷冻前后六种酶活性的变化及ATP含量结果显示，经过超低温冷冻后，精子内SOD、GSH-PX和CAT三种酶及ATP含量均有显著性降低（P<0.05）。精浆中酶活力升高，除GR和CAT外，其余酶活性均差异显著（P<0.05）。【结论】长期超低温冷冻对鞍带石斑鱼精浆和精子酶活性、精子活力及精子超微结构均具有较显著影响，【意义】研究结果为鱼类精子冷冻损伤机理研究积累了丰富的数据，为鱼类精子长期冷冻保存提供了技术参考和评价指标。     

鱼类冷冻精子结构变异的电子显微镜研究

<正> 引言液氮冻结保存鱼类精子,国内外虽已有成功的报道,但现有的冷冻技术水平,还无法克服精子的活力随操作顺序而逐趋下降的缺点,致使受精率、孵化率不够稳定,研究中也尚有许多不明之处,因此造成此项技术还未能在生产上迅速推广应用。若干年来,国内外都较侧重于冷冻技术(稀释液成分、冻结和解冻方法)方面的研究,而对冷冻中各个环节引起精子正常结构形态的变异,从而使复苏率、受精率受到影响的报告,迄今还未见有发表。我们用电子显微镜着重观察     

Effects of Broodstock Age and Sperm Collection Time over a Natural Spawning Period on Sperm Cryopreservation in Farmed Greenlip Abalone,Haliotis laevigata

This study investigated the effects of broodstock age (2‐ and 3‐yr‐old), and sperm collection time at the beginning, middle, and end of a natural spawning period, on the ability of sperm to tolerate cryopreservation in farmed greenlip abalone, Haliotis laevigata. The quality of sperm was assessed by motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential (MMP), and acrosome integrity (AI). The sperm collected at the middle of a natural spawning period had significantly higher quality than those collected at the beginning and the end of the spawning period in terms of plasma membrane integrity, MMP, and AI of fresh and post‐thaw sperm, post‐thaw sperm motility, and fertilization rate. No significant difference was found between 2‐ and 3‐yr‐old animals in most quality parameters evaluated. The results suggested that sperm collected during the middle of the spawning season should be used for cryopreservation. The efficiency of genetic improvement programs can be further enhanced by using sperm collected from 2‐yr‐old abalone which has a similar ability to tolerate cryopreservation as 3‐yr‐old counterparts.     

Sperm quality and cryopreservation of Brazilian freshwater fish species: a review

The Brazilian freshwater fish diversity is the richest in the world. Only 0.7% of all Brazilian species have had any aspect of their sperm biology addressed up to this date. The majority of the fish species described in this review migrate during the spawning season (a phenomenon known as piracema). Urbanization, pollution, hydroelectric dams and deforestation are some of the causes of stock depletion or even local extinction of some of these species. The knowledge concerning sperm quality and minimum sperm:egg ratio is important to maximize the use of males without reducing hatching rates. Furthermore, sperm cryopreservation and gene banking can guarantee the conservation of genetic diversity and development of adequate breeding programs of native fish species. In this review, we present and evaluate the existing information on Brazilian fish species that have been subject to sperm quality and cryopreservation studies. The following parameters were evaluated: volume of extractable sperm, sperm motility, sperm concentration, freezing media, freezing methods, and post-thaw sperm quality. Although the existing protocols yield relatively high post-thaw motility and fertilization rates, the use of cryopreserved sperm in routine hatchery production is still limited in Brazil.     

Toxic effects of mercury chloride on silver catfish (Rhamdia quelen) spermatozoa

Heavy metals are highly toxic elements that are present in the environment, especially in water. Mercury chloride (HgCl₂) stands out among these compounds because of its strong ability to induce damage to any tissue with which it comes into contact. The gametes of spawning aquatic animals, such as fish, are susceptible to such damage. Thus, our objective was to evaluate the toxic potential of HgCl₂ in the capacitation and activation of Rhamdia quelen sperm. Semen was collected from seven males and activated in 58 mM sodium chloride (NaCl) containing 0 (control), 4^?10, 7^?10, 7^?9, and 7^?8 M HgCl₂. The evaluated variables included motility, vigor, motility time, morphology, membrane integrity, membrane fluidity, mitochondrial functionality, production of reactive oxygen species (ROS), and DNA fragmentation. All evaluated HgCl₂ concentrations increased primary pathologies and reduced motility, vigor and motility time. Damage to membrane integrity and fluidity began occurring at a concentration of 7^?10 M HgCl₂. These results indicate that HgCl₂ has a toxic effect on different sites of fish spermatozoa and that sperm motility decreases after exposure to HgCl₂, impairing sperm capacitation and activation.     

Subjective and objective assessment of fish sperm motility: when the technique and technicians matter

Fish sperm motility is nowadays considered the best sperm quality biomarker in fish, and can be evaluated both by subjective and computerized methods. With the aim to compare the precision and accuracy of both techniques, fish sperm samples were assessed by subjective methods and by a computer-assisted sperm analysis (CASA-Mot) system, and simultaneously by three different technicians with different degrees of expertise on the sperm quality analysis. Statistical dispersion parameters (CV, coefficient of variation; and RG, range) were estimated in order to determine the precision and accuracy of the techniques and the influence of laboratory staff on sperm motion assessments. Concerning precision, there were not much significant differences between the technical support staff (high, medium, and low experimented technician), and statistical dispersion parameters were quite similar between them independent of the technique used and the sperm motility class analyzed. However, concerning accuracy, experimented technician reported subjective motility values very closed to the values provided by the CASA-Mot system, only 10 percentage points away from the data provided by a CASA-Mot system. However, medium and low experimented technicians often overestimate the CASA-Mot values, and amplitudes up to 30 percentage points were detected in several sperm assessments. To sum up, both the technique (subjective or objective) and the technician (degree of expertise) became key factors in order to reach accurate motility estimations, so the use of both qualified staff and novel CASA-Mot systems seems to be a critical requirement for obtaining satisfying results in fish species with similar motility patterns.     

Vitrification of sperm from marine fish: effect on motility and membrane integrity

Our goal was to develop a standardized approach for sperm vitrification of marine fish that can be applied generally in aquatic species. The objectives were to: (i) estimate acute toxicity of cryoprotectants over a range of concentrations; (ii) evaluate the properties of vitrification solutions (VS); (iii) evaluate different thawing solutions and (iv) evaluate sperm quality after thawing by examination of motility and membrane integrity. Sperm were collected from red snapper (Lutjanus campechanus), spotted seatrout (Cynoscion nebulosus) and red drum (Sciaenops ocellatus). A total of 29 combinations of cryoprotectants were evaluated for toxicity and glass formation. Samples were loaded onto 10‐μL polystyrene loops and plunged into liquid nitrogen. There was a significant difference (P < 0.05) in post‐thaw motility among VS and among species when using the same VS. The sperm in VS of 15% DMSO + 15% ethylene glycol + 10% glycerol + 1% X‐1000^? + 1% Z‐1000^? had an average post‐thaw motility of 58% and membrane integrity of 19% for spotted seatrout, 38% and 9% for red snapper, and 30% and 19% for red drum. Adaptations by marine fish to higher osmotic pressures could explain the survival in the high cryoprotectant concentrations. Vitrification offers an alternative to conventional cryopreservation.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage

Spermatozoa and seminal plasma obtained from rainbow trout and whitefish were analyzed in respect to their aspartate aminotransferase (AspAT) and alkaline phosphatase activities. In particular, the experiments characterized AspAT optimum pH, optimization of assay conditions and action of coenzyme, pyridoxal 5-phosphate (vitamin B₆). The effect of short-term semen storage at 0°C on biochemical indicators and fertilization rate was examined in both species. The concentrations of reduced and oxidized ascorbic acid in seminal plasma of both species were several folds higher than in spermatozoa and blood plasma of fish. Highly significant correlations were found for both species between AspAT activity (sperm or seminal plasma) and fertilization rate (% of eyed-stage or hatched embryos). For rainbow trout, highly significant correlations were found between sperm concentration, motility and fertilization rate. These results suggest that several biochemical indicators of seminal plasma can be used as measures of sperm quality of fish. Some common biochemical parameters for fish and mammal's semen provide evidence for using fish sperm as a model in biomedical research.     

Electric ultrafreezer (− 150 °C) as an alternative for zebrafish sperm cryopreservation and storage

Zebrafish sperm cryopreservation is a fundamental methodology to manage and back-up valuable genetic resources like transgenic and mutant strains. Cryopreservation usually requires liquid nitrogen for storage, which is expensive and hazardous. Our objective was to evaluate if electric ultrafreezers (??150 °C) are a viable alternative for zebrafish sperm storage. Zebrafish sperm was cryopreserved in the same conditions (??20 °C/min), stored either in liquid nitrogen or in an ultrafreezer, and thawed after 1 week, 1 month, and 3 months. Sperm motility, membrane integrity, and fertilization ability were assessed. There were no significant differences in motility and hatching rate throughout storage time. Additionally, we aimed at understanding if cryopreservation directly in an ultrafreezer (??66 °C/min) could improve post-thaw sperm quality. Freezing at ??20 °C/min was performed as before, and compared to samples cryopreserved with a fast cooling rate by placing directly in an ultrafreezer (??66 °C/min). Sperm quality was assessed according to motility, viability, DNA fragmentation, and apoptosis (annexin V). The ??66 °C/min cooling rate showed significantly higher membrane and DNA integrity, and lower number of cells in late apoptosis in comparison to the other treatments. This study showed that zebrafish sperm cryopreservation and storage in an ultrafreezer system is possible and a fast cooling rate directly in ultrafreezer improves post-thaw sperm quality.     

The effects of osmolality on sperm quality in <Emphasis Type="Italic">Jenynsia multidentata</Emphasis> (Cyprinodontiformes: Anablepidae)

Sperm quality tests on fish are classically used for evaluating cryopreservation procedures, and they are also promising to assess aquatic toxicity and biomarkers of xenobiotic effects on reproduction. Osmotic shock from the storage medium is one of the main factors affecting sperm quality during evaluation. Thus, the objective of this study was to evaluate the effects of different osmolalities (240–460 mOsm/kg) for at least 4 days on the sperm quality parameters of the viviparous fish Jenynsia multidentata. The level of significance was (P < 0.05). The plasma osmolality of J. multidentata is 326 ± 3.9 mOsm/kg. The motility of fresh semen was higher in osmolalities of 280 and 300 mOsm/kg but did not differ between osmolalities from 240 to 320 mOsm/kg. Above 380 mOsm/kg, the motility observed was 0 %. Over the time period studied motility increased with increasing osmolality, and the most constant and long-lasting rates were between 300 and 320 mOsm/kg. On the 4th day of evaluation, higher membrane integrity rates were observed between 280 and 360 mOsm/kg, higher mitochondrial membrane potential was observed between 300 and 460 mOsm/kg, and higher DNA integrity rates were observed between 260 and 380 mOsm/kg. Moreover, osmolalities ≥460 and ≤240 resulted in the lowest motility and DNA integrity levels. Over 4 days, the plasma membrane integrity was significantly lower at ≤260 and ≥400 mOsm/kg, and the mitochondrial membrane potential was significantly lower only in osmolalities ≤240 mOsm/kg. Therefore, we conclude that for sperm quality preservation in J. multidentata, an osmolality of 300–320 mOsm/kg of the most suitable diluent is necessary. Furthermore, we conclude that the storage of sperm in a hyposmotic (<260 mOsm/kg) or hyperosmotic (>400 mOsm/kg) solution affects not only motility but also other sperm quality parameters.     

Effect of water temperature on the physiology of fish spermatozoon function: a brief review

Motility is a key factor in function of the spermatozoon and determines semen quality and fertilizing capacity. Effective motility occurs when sperm is diluted in a swimming solution, the adequacy of which is determined by factors varying according to fish species. Spermatozoon motility rate and velocity, as well as duration of the motility period, are influenced by the temperature of the water in which broodfish are held. Increase in temperature of swimming medium beyond the optimal increases cell metabolism, leading to an increase in velocity with rapid depletion of energy resources, promoting early cessation of movement. The aim of this review was to discuss current information on the influence of temperature on quantitative spermatozoon properties, which could affect sperm function. Our findings provide a greater understanding of fish sperm physiology and a biological foundation for the further development of spermatozoon motility investigations as well as reproduction technologies.